The cardiac ryanodine receptor (ryanodine receptor 2, RyR2) Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane plays a central role in cardiac excitation—contraction coupling (Bers, J Mol Cell Cardiol, 2004; 37:417-429). During the past 10 years, diastolic Ca2+ leak through dysfunctional RyR2 has been recognized as an important factor contributing to altered Ca2+ homeostasis and arrhythmias in heart failure (HF). Evidence from several reports shows that RyR2 abnormality in HF causes increased diastolic Ca2+ leak, leading to contractile and relaxation dysfunction (Yano et al., Circulation, 2000; 102:2131-2136; Ai et al., Circ Res, 2005; 97:1314-1322; Wehrens et al., PNAS USA, 2006; 103:511-518). Furthermore, the abnormal Ca2+ leak through RyR2 provides a substrate for delayed after depolarization that leads to lethal arrhythmias (Wehrens et al., Cell, 2003; 113:829-840). The connection between cardiac dysfunction and RyR2 leak is extensively discussed in Bers D M (Annu Rev Physiol, 2014; 76:107-127)
One leading hypothesis explains the RyR2 dysfunction in HF and lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), by structural RyR2 changes that result in defective interaction (or zipping) between the N-terminal (N: 0-600) and the central (C: 2000-2500) domains (Yamamoto et al., Biochem Biophys Res Commun, 2002; 291:1102-1108). According to this concept, in the resting state, the N-terminal and central RyR2 domains interact with each other to act as a regulatory switch that influences RyR channel gating. This tight interdomain interaction, termed domain zipping, seems to stabilize the closed channel. Weakening of these interdomain interactions may be caused by mutations either in the N-terminal or central regions of RyR2 (Uchinoumi et al., Circ Res, 2010; 106:1413-1424) or via competition by peptides derived from these 2 domains (domain unzipping), (Ikemoto, Front Biosci, 2002; 7:d671-d683; Ikemoto in “Ryanodine Receptors: Structure, Function and Dysfunction in Clinical Disease,” New York, N.Y.: Springer; 2004:53-65; Yamamoto et al., J Biol Chem, 2000; 275:11618-11625) resulting in an increased opening probability of the RyR2 and leakiness of Ca2+. Domain peptide corresponding to RyR2 residues 2460-2495 (DPc10) is a synthetic peptide corresponding to a 36-residue stretch of the central domain (Gly2460-Pro2495) of RyR2 (Yamamoto et al., Biochem Biophys Res Commun, 2002; 291:1102-1108). It has been shown that DPc10 can specifically and directly associate with the N-terminal domain, (Oda et al., Circulation, 2005; 111:3400-3410; Tateishi et al., Cardiovasc Res, 2009; 81:536-545) and thus can compete with its zipping to the central domain, and that the N-domain/DPc10 association can destabilize RyR2 (via domain unzipping) to increase Ca2+ leakiness (Oda et al., Circulation, 2005; 111:3400-3410). A single point mutation in DPc10 (R2474S) prevents all DPc10 effects, and a related human RyR2 mutation is associated with CPVT and RyR2 leakiness.
Tateishi et al. (Tateishi et al., Cardiovasc Res, 2009; 81:536-545) reported that a domain peptide (residues 163-195 of the N-terminal RyR2 domain, DP163-195) also induced Ca2+ leak from SR, presumably because it binds to the central domain and competes with the N-terminal/central zipping.
The FK506-binding proteins FKBP12 and FKBP12.6 are expressed in cardiac myocytes and can form tight complexes with RyR at a stoichiometry of 4 FKBPs per tetrameric RyR channel (Bers, J Mol Cell Cardiol, 2004; 37:417-429). As such, these FKBP isoforms are considered important RyR2 subunits and have been reported to promote the closed channel state, but this role is controversial in myocytes from normal rat hearts (Bers, Circ Res, 2012; 110:796-799). FKBP12 does not significantly alter Ca2+ sparks, whereas FKBP12.6 is slightly inhibitory, PKA-dependent RyR2 phosphorylation does not alter FKBP binding, and only a small fraction of RyR2 in native myocytes is FKBP12.6-bound (Guo et al., Circ Res. 2010; 106:1743-1752). Two previous studies in which RyR2 was treated with domain peptides to mimic pathological Ca2+ leakage found no direct effect of DPc10 on FKBP12.6 coimmunoprecipitation with RyR2 (Oda et al., Circulation, 2005; 111:3400-3410; Tateishi et al., Cardiovasc Res, 2009; 81:536-545). It is unknown whether FKBP12.6 influences binding of DPc10 to RyR2 or the ensuing increased Ca2+ leakage.
Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that binds to the RyR2 and modulates its channel function (Yamaguchi et al., J Biol Chem, 2003; 278:23480-23486). Binding of CaM within the cytosolic domain of RyR2 (at a site partly formed by residues 3583-3603) inhibits channel activity both at diastolic and at elevated [Ca2+] (Fruen et al., Am J Physiol, Cell Physiol, 2000; 279:C724-C733; Balshaw et al., J Membr Biol, 2002; 185:1-8). This indicates that CaM stabilizes the closed state of RyR2 in the resting state (Guo et al., Circ Res. 2006; 99:398-406). Interestingly, concurrent addition of a high concentration of CaM with DPc10 in wild-type cardiomyocytes reduced the Ca2+ spark frequency (CaSpF) (calcium spark frequency) compared with addition of DPc10 alone. Furthermore, myocytes carrying a CPVT-linked RyR2 mutation (where β-adrenergic stimulation activates SR Ca leak) have defective interdomain interaction and reduced CaM binding to the RyR2 vs wild-type myocytes (Xu et al., Biochem Biophys Res Commun, 2010; 394:660-666). In addition, Ono et al. (Ono et al., Cardiovasc Res, 2010; 87:609-617) also reported that the CaM-binding affinity to RyR2 in HF is significantly reduced compared with that of normal RyR2. Treatment of wild-type myocytes with DPc10 also inhibited CaM binding at the Z-line in the CPVT mutants (Xu et al., Biochem Biophys Res Commun, 2010; 394:660-666).